The complete mitogenome of the pond wolf spider Pardosa pseudoannulata, with phylogenetic implications for the Lycosidae

Abstract The pond wolf spider Pardosa pseudoannulata Bösenberg & Strand, 1906 (Araneae: Lycosidae) is an important predator of agricultural pests in southern, eastern and southeastern Asia. Here, we report the complete mitogenome of this spider reconstructed from Illumina sequencing data. The circular mitogenome length is 14,533 bp with the nucleotide composition A (33.3%), C (8.2%), G (15.2%), and T (43.3%). The P. pseudoannulata mitogenome comprises 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. Phylogenetic analyses of Lycosidae mitogenomes supported the monophyly of the subfamily Pardosinae and the two genera Pardosa and Alopecosa, and indicated the polyphyly of the subfamily Lycosinae and the paraphyly of its type genus Lycosa. In this study, P. pseudoannulata is the closest relative to P. pusiola. These results provide useful genetic information for future studies on the diversity, phylogeny, and evolution for wolf spiders.


Introduction
Globally, spiders are composed of 135 families, 4,376 genera, and 51,993 species (World Spider Catalog 2024).They are important predators for the insect pests of agriculture, forestry, fruit trees, and other economically important plants (Yang et al. 2018;Li et al. 2021).The wolf spider familar, Lycosidae is the fifth largest group in the order Araneae with 133 genera, and 2,478 species (World Spider Catalog 2024).Pardosa is the largest genus in the family with 532 species, and the spiders of this genus distribute worldwide (World Spider Catalog 2024).The pond wolf spider P. pseudoannulata B€ osenberg & Strand, 1906 is an Asian species from South, Southeast and East Asia (Song et al. 1999;World Spider Catalog 2024).It preys on many species of insect pests that are of agricultural importance and, as a consequence, this spider has been looked as a possible biological control agent (Li et al. 2017).The increasing interest in the potential of spider venom as a tool for pest control have prompted a detailed study on this spider (Huang et al. 2018).In this study, we sequenced a P. pseudoannulata mitogenome, and analyzed the phylogenetic relationships of the major subfamilies and genera within Lycosidae using mitogenomes.These results will be useful in the future studies for phylogenetics, population genetics, and biogeography within Araneae.

DNA extraction and sequencing
Total genomic DNA was extracted from the cephalothorax and legs of the male sample using the modified cetyltrimethyl ammonium bromide (CTAB) method (Shahjahan et al. 1995).The Illumina Novoseq 6000 sequencing platform was used for sequencing, and the Illumina PE150 library was constructed at Personalbio Biotechnology Company (Shanghai, China).
In order to understand the phylogenetic status of P. pseudoannulata and the phylogeny of major subfamilies and genera within Lycosidae, we downloaded the 16 lycosid mitogenomes from the NCBI database.The mitogenomes of Pisaura bicornis (GenBank accession number: MN296112) and Dolomedes angustivirgatus (GenBank accession number: NC031355) of Pisauridae were selected as outgroups.Maximum-likelihood (ML) method in W-IQ-TREE 2.0 (Hoang et al. 2018, 1000 ultrafast bootstrap) was used to reconstruct phylogenetic tree using the 13 PCGs.Sequences were aligned using MAFFT 7 (http://mafft.cbrc.jp/alignment/server/)with the L-INS-i method.To detect and exclude ambiguously aligned regions, alignments of the 13 PCGs were processed with the program trimAl v1.3 (Capella-Guti� errez et al. 2009).We inferred ML topologies based on the partitioning strategy.For each gene partition, the best-fitting substitution model was estimated simultaneously using the greedy algorithm in ModelFinder (Kalyaanamoorthy et al. 2017).We used the Bayesian information criterion and the FreeRate heterogeneity to select the best-fitting substitution model for each gene partition ).The perturbation strength (p) and the number of iterations since the last best tree found (c) were set to 0.3 and 1000, respectively.

Phylogenetic indications
The phylogeny of Lycosidae including 17 species of six genera was reconstructed using the 13 PCGs (Figure 3).Our result indicates that P. pseudoannulata and Wadicosa fidelis belong to the subfamily Pardosinae, and supported the monophyly of Pardosinae.Both Pardosa and Alopecosa are monophyletic.The genus Lycosa is paraphyletic.Alopecosa and Lycosa species clustered together, and Lycosinae is a polyphyletic group.The genus Halocosa may not belong to the subfamily Lycosinae.

Discussion and conclusions
Lycosid spiders constitute an extensive and diverse branch of the order Araneae.Whereas the mitogenomes of only 16 species of wolf spiders have been determined.Our results showed that the mitogenomic structure of the pond wolf spider P. pseudoannulata was similar to that of other lycosid species (Ding et al. 2022;Ye et al. 2023;Yi et al. 2023).The reconstructed phylogenetic tree illuminated the positions of P. pseudoannulata and W. fidelis in subfamily Pardosinae of Lycosidae, and the phylogenetic relationships between 17 species of the six lycosid genera, and indicated the subfamily Lycosinae and the genus Lycosa were both non-monophyletic.Azarkina and Trilikauskas (2019) assigned Halocosa to the subfamily Lycosinae based on the lateromedian origin of the embolus that is situated in a shallow and wide depression.However, our molecular phylogeny indicated that Halocosa hatanensis may not be a species of Lycosinae.The results of this study will contribute to future studies on phylogenetics, population genetics, and biogeography within the family Lycosidae.

Figure 2 .
Figure 2. The circular map of the P. pseudoannulata mitogenome.The inner ring indicates the GC content.

Figure 3 .
Figure 3.The ML phylogenetic tree reconstructed using the 13 PCGs (the sequenced P. pseudoannulata mitogenome by this study marked with asterisk).Pisaura bicornis and Dolomedes angustivirgatus (Pisauridae) were used as outgroups.The scale-bar indicates 0.09 substitutions per site.The numbers to the left of each node denote the ultrafast bootstrap value for ML analysis.The following sequences were used: ON411610 (Li et al. 2022), ON411611 (Li et al. 2022), ON419104 (Li et al. 2022), MW776434 (Li et al. 2022), and OK032619 (Ye et al. 2022).